Four New Sesquiterpenoids from the Rice Fermentation of Antrodiella albocinnamomea

Albocimea B-E (1–4), four new sesquiterpenoids, and four known compounds, steperoxide A (5), dankasterone (6), 1H-indole-3-carboxylic acid (7), and (+)-formylanserinone B (8), were isolated from the rice fermentation of the fungus Antrodiella albocinnamomea. The structures of new compounds were elucidated by comprehensive spectroscopic techniques, the planar structures of new compounds were determined by comprehensive spectroscopic techniques, and their absolute configurations were confirmed via gauge-independent atomic orbital calculations (GIAO), calculation of the electronic circular dichroism (ECD), and optical rotation (OR). These were determined by spectroscopic data analysis.

In the ROESY spectrum, only weak correlations between H3-14 and H2-1 can be observed ( Figure S9, see details in the Supplementary Materials). In order to further determine the relative configurations of 1, gauge-independent atomic orbital calculations (GIAO), 13 C NMR calculations, and DP4+ analysis were performed. Compound 1 has two chiral centers, which have four possible configurations. The 13 C-NMR of four possible structures was calculated at the mPW1PW91/6-31 G (d) level using the GIAO method. Comparison of the 13 C chemical shifts obtained revealed that the calculated chemical shifts of 1a and 1c (Table S3 and Figure S35, see details in the Supplementary Materials) were closest to the experimental values. Therefore, the relative configurations of 1 were designated. Finally, the absolute configurations of 1 were assigned as 6R,7S by comparison of the experimental and calculated ECD ( Figure S1). As shown in Figure S1, the calculated curve for 6R,7S matches well with that of the experimental ECD curve of compound 1. Thus, the structure of albocimea B (1) was determined as depicted.
Compound 2 was obtained as a white amorphous powder, based on a Na + adduct at m/z 236.1408 [M+Na] + (calculated for 236.1412) via HR-ESI-MS, the molecular formula of

Results and Discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula of C 14 (Table 1) revealed resonances for three methyl protons at δ H 2.20, 1.14, and 0.98 (each 3H, s), and revealed that those methyl groups were located at quaternary carbons. The 13 C NMR and DEPT spectra (Table 1) revealed the existence of 14 carbon signals for three methyls, four methylenes, one olefinic methine (δ C 121.3), and six quaternary carbons (including two ketone carbonyls at δ C 194.7 and 213.1, one oxygenated at δ C 91.5, and one olefinic at δ C 145.0). These spectroscopic data revealed that 1 should be a dicyclic norsesquiterpenoid. The 1 H-1 H COSY spectrum ( Figure 2) of 1 established the partial structures of H 1 -4/H 2 -5 and H 2 -9/H 2 -10. The HMBC correlations ( Figure 2) from H 3 -12 (δ H 1.14) to C-13, C-11, C-10, and C-6, indicated C-12 and C-13 were located on C-11, and C-10 and C-6 were connected by C-11. The HMBC correlations ( Figure 2) from H 3 -14 (δ H 2.20) to C-7, C-8, and C-6, and H 2 -9 to C-7 indicated C-8 and C-6 were connected by C-7. Beside this, C-5 and C-1 were connected by C-6 from the 1 H-1 H COSY correlation of H 1 -4/H 2 -5, and HMBC correlations from H 2 -5 and H 2 -1 to C-6, C-7, and C-11, and so established a 6,6-bicylic skeleton via a C-6 spiro carbon. These data of 1 are closely related to those of antroalbol H [3]. The difference between them is the presence of a double bond group at C-3 and C-4 in compound 1. This assignment was in accordance with the HMBC correlations ( Figure 2) from H-4 (δ H 6.03 br s) to C-5, C-6, and C-2, and H 2 -5 to δ C 145.0 (C-3).
In the ROESY spectrum, only weak correlations between H 3 -14 and H 2 -1 can be observed ( Figure S9, see details in the Supplementary Materials). In order to further determine the relative configurations of 1, gauge-independent atomic orbital calculations (GIAO), 13 C NMR calculations, and DP4+ analysis were performed. Compound 1 has two chiral centers, which have four possible configurations. The 13 C-NMR of four possible structures was calculated at the mPW1PW91/6-31 G (d) level using the GIAO method. Comparison of the 13 C chemical shifts obtained revealed that the calculated chemical shifts of 1a and 1c (Table S3 and Figure S35, see details in the Supplementary Materials) were closest to the experimental values. Therefore, the relative configurations of 1 were designated. Finally, the absolute configurations of 1 were assigned as 6R,7S by comparison of the experimental and calculated ECD ( Figure S1). As shown in Figure S1, the calculated curve for 6R,7S matches well with that of the experimental ECD curve of compound 1. Thus, the structure of albocimea B (1) was determined as depicted.  13 C NMR (150 MHz) data for compounds 1-3 in CDCl 3 and 4 in CD 3 OD (δ in ppm, J in Hz).
No.  Because the isolated compound materials are limited, only the ones with sufficient amount could be tested for bacteriostatic test. Therefore, compounds 2 and 6 were evaluated for antibacterial activity with the Kirby-Bauer test. The results showed that both had no significant inhibitory activity against Psecdomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Monilia albican.
The correlation of H 3 -13 and H 2 -4 is very weak in the ROESY spectrum ( Figure S17). In order to further determine the relative configurations of 2, we adopted the same method as in determining the configurations of compound 1. Because compound 2 has two chiral centers, it has four possible configurations. 13 C-NMR calculations of these four possible configurations were carried out at the mPW1PW91/6-31 G (d) level using the GIAO method. By comparison of 13 C chemical shifts, the relative configurations of 2 can be determined. In order to determine its absolute configuration, the optical rotation (OR) value of configuration (5S,6S)-2 was calculated; this value is −9.75. Compared with the experimental OR value of compound 2 (−10.2), the absolute configuration of 2 was finally determined as 5S,6S. Thus, the structure of albocimea C (2) was assigned as depicted.
The CEs in the CD spectrum are not obvious for 3; the absolute configuration of this compound was further investigated by comparison of its experimental OR value with those calculated for (2S)-3. The calculation results show that the calculated OR value of (2S)-3 is +3.01, while the comparative experimental OR value is −2.92. Therefore, it is suggested that the absolute configuration of 3 is opposite to the calculated configuration. Finally, the absolute configuration of 3 was determined as 2R. Thus, the structure of albocimea D (3) was assigned as depicted.
The correlations observed in the ROESY spectrum ( Figure S34) of 4 were insufficient for determining its relative configuration. Because compound 4 has three chiral centers, it has eight possible configurations. 13 C-NMR calculations of eight of these possible configurations were carried out at the mPW1PW91/6-31 G (d) level using the GIAO method. The comparison of the 13 C chemical shifts obtained revealed that the calculated chemical shifts of an enantiomer pair configuration 4a and 4e (Table S3 and Figure S35) are the closest to the experimental values. Finally, the absolute configurations of 4 were assigned as 5R,9S,13S by comparison with the experimental and calculated ECD ( Figure  S26); the calculated curve for 5R,9S,13S matches well with that of the experimental ECD curve of 4. Thus, compound 4 was established to be albocimea E.
Because the isolated compound materials are limited, only the ones with sufficient amount could be tested for bacteriostatic test. Therefore, compounds 2 and 6 were evaluated for antibacterial activity with the Kirby-Bauer test. The results showed that both had no significant inhibitory activity against Psecdomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Monilia albican.
In conclusion, four previously undescribed sesquiterpenoids (1-4) and four known compounds (5)(6)(7)(8) were acquired from the rice fermentation of the fungus A. albocinnamomea. The structures of these compounds were characterized using spectroscopic data. The antibacterial activity test of compounds 2 and 6 showed that they have no significant antibacterial activity.

General Experimental Procedures
Optical rotations were taken on a JASCO P-1020 polarimeter. IR spectra were obtained on a Bruker Tensor 27 spectrometer with KBr pellets. NMR spectra were measured on a Bruker Avance III 600 MHz spectrometer with TMS as the internal standard. Mass spectra were recorded with an APIQSTAR time-of-flight spectrometer. CD spectra were recorded on an Applied Photophysics spectrometer. Silica gel (200-300 mesh), Sephadex LH-20, and Rp-C 18 were used for column chromatography (CC). Thin-layer chromatography (TLC) experiments were performed on a silica gel GF 254 pre-coated plate. Fractions were monitored by TLC, and spots were visualized by spraying with 15% H 2 SO 4 in ethanol.

Fungal Material and Cultivation Condition
A. albocinnamomea was purchased from the China Institute of Microbiology. A voucher specimen (No. Yang20181012) was deposited at the Faculty of Life Science and Technology, Kunming University of Science and Technology. A rice medium was used to ferment the strain. The culture of the strain was divided into two steps. Firstly, the fungal strain was cultured in potato dextrose agar (PDA) medium at 24 • C, and the seed solution was obtained after 7 days of culture. Next, a rice medium was used for large-scale fermentation. The culture medium consisted of rice and water at a ratio of 1:1.4. When preparing the culture medium, 71 g of rice and 100 mL of water were put into 480 mL fermentation bottles. A total of 300 bottles were prepared. They were put into a high-pressure steam sterilization pot and sterilized at 121 • C for 30 min. The seed solution obtained before was divided into small parts, put into the prepared rice medium and incubated at room temperature for 45 days.

Bacterial Strain
The strain of Peseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Monilia albican were purchased from the Nanjing Bianzhen Biotechnology Co., Ltd (Nanjing, China) and deposited at the Faculty of Life Science and Technology, Kunming University of Science and Technology.